WO2012083240A2 - Puce d'oligonucléotides pour le typage tissulaire - Google Patents

Puce d'oligonucléotides pour le typage tissulaire Download PDF

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WO2012083240A2
WO2012083240A2 PCT/US2011/065624 US2011065624W WO2012083240A2 WO 2012083240 A2 WO2012083240 A2 WO 2012083240A2 US 2011065624 W US2011065624 W US 2011065624W WO 2012083240 A2 WO2012083240 A2 WO 2012083240A2
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drb1
hla
dpb1
dqb1
oligonucleotide
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WO2012083240A3 (fr
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Ellis L. Reinherz
Vladimir Brusic
Guanglan Zhang
Derin Keskin
David Deluca
Honghuang Lin
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Dana-Farber Cancer Institute, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • This invention relates to tissue typing using an oligonucleotide-based microarray. More particularly, the HLA arrays are high resolution arrays useful for diagnostic evaluations and determining donor/recipient transplant compatibility.
  • HLA variation is a crucial determinant of transplant rejection, susceptibility to a large number of infectious and autoimmune diseases, and cancer.
  • the limiting factor in large- scale genetic analysis of, for example, transplant populations has been methodologic and directly involves the technical ability to accurately define the alleles of highly polymorphic HLA genes in a cost-effective and efficient manner.
  • sequencing and array-based methods has allowed alleles to be determined with accuracy, large-scale efforts in genetic analysis of transplant populations are hampered by the cost and inefficiency of available methods.
  • HLA arrays are inefficient, because resolution of ambiguities is not guaranteed, and multiple arrays are needed to conduct a complete analysis of all HLA genes.
  • Exemplary limitations of currently available HLA arrays include for example, that they are suitable for low-to-medium density genotyping but not for high- density genotyping; they do not have a complete set of probes (i.e. capture oligonucleotides that recognize target HLA encoding nucleic acids), but rather have only a selection of "informative probes", thereby making deconvolution of ambiguities difficult or impossible; they do not cover all currently known HLA alleles from each group; and they do not encode a complete set of classical and non-classical HLA loci.
  • the invention provides a method for human leukocyte antigen (HLA) tissue typing, said method comprising: (a) contacting a cDNA- or cRNA- containing sample under hybridization conditions with a plurality of capture oligonucleotides specific for HLA polypeptides, wherein said hybridization conditions facilitate hybridization of a subset of capture oligonucleotides to complementary sequences present in the cDNA or cRNA; (b) detecting a hybridization pattern for said cDNA or cRNA; and (c) assigning to the sample, based on the hybridization pattern, an HLA tissue type; wherein the capture oligonucleotides are from about 17 to about 60 nucleotides in length and each capture oligonucleotide with respect to its exact complement has a melting temperature of about 64 degrees Celsius; wherein said capture oligonucleotides comprise subsets of oligonucleotides that collectively target classical HLA polypeptide-en
  • step (a) said cDNA or cRNA was detectably labeled during the making, and said detecting step (b) comprises detecting said detectably labeled cDNA or cRNA.
  • the detecting step (d) comprises the use of labeled detection probes.
  • the capture oligonucleotides comprise subsets of oligonucleotides that collectively target all known classical HLA polypeptide- encoding nucleic acids.
  • the capture oligonucleotides are immobilized on a substrate.
  • said classical HLA polypeptide-encoding nucleic acids encode HLA polypeptides selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
  • said capture oligonucleotides further comprise a plurality of oligonucleotide subsets that collectively targets non-classical HLA polypeptide-encoding nucleic acids ("non-classical HLA oligo subsets”), each non-classical HLA oligo subset targeting a different non-classical HLA polypeptide-encoding nucleic acid; wherein each of said non-classical HLA oligo subsets comprises a set of overlapping oligonucleotides that cover every single nucleotide position in the mRNA sequences coding for the non-classical HLA polypeptides from 5' to 3' and are sequentially shifted by 1-5 nucleotides from the 5' end of the preceding overlapping oligonucleotide.
  • the plurality of oligonucleotide subsets that collectively targets non-classical HLA polypeptide-encoding nucleic acids collectively target all known non-classical HLA polypeptide-encoding
  • said non-classical HLA polypeptide-encoding nucleic acids encode HLA polypeptides selected from the group consisting of HLA-E, HLA-F, HLA- G, DM, DO and MIC.
  • said capture oligonucleotides further comprise a plurality of oligonucleotide subsets that collectively targets nucleic acids encoding accessory molecules important in HLA-linked peptide presentation and/or processing ("accessory molecule oligo subsets"), and said method further comprises the step of assigning to the sample, based on the hybridization pattern, an accessory molecule phenotype; wherein each of said accessory molecule oligo subsets comprises a set of overlapping oligonucleotides that cover every single nucleotide position in the mRNA sequences coding for the accessory molecules from 5' to 3' and are sequentially shifted by 1-5 nucleotides from the 5' end of the preceding overlapping oligonucleotide.
  • said accessory molecules are selected from the group consisting of LMP2, LMP7, LMP10, tripeptidyl peptidase II (TPPII), bleomycin hydrolase (BLMH), leucine aminopeptidase 3 (LAP3), transporter associated with antigen processing (TAP) 1, TAP2, 2-microglobulin, TAP binding protein (tapasin), calnexin (CANX), calreticulin (CALR), protein disulfide isomerase family A member 2 (PDIA2), protein disulfide isomerase family A member 3 (PDIA3), ERp57, endoplasmic reticulum aminopeptidase (ERAP) 1, ERAP2, proteasome (prosome macropain) subunit althap (PSMA) type I (PSMA1), PSMA2, PSMA3, PSMA4, PSMA5, PSMA6, PSMA7, PSMA8, proteasome (prosome macropain) subunit beta (PSMB) type 1 (PSMB1), PSMB2, PSMB3, PS
  • said capture oligonucleotides further comprise a plurality of oligonucleotide subsets targeting killer-cell immunoglobulin-like receptor (KIR) polypeptide-encoding nucleic acids ("KIR oligo subsets”), and said method further comprises the step of assigning to the sample, based on the hybridization pattern, a KIR polypeptide phenotype; wherein each of said KIR oligo subsets comprises a set of overlapping oligonucleotides that cover every single nucleotide position in the mRNA sequences coding for the KIR polypeptides from 5' to 3' and are sequentially shifted by 1-5 nucleotides from the 5' end of the preceding overlapping oligonucleotide.
  • KIR oligo subsets targeting killer-cell immunoglobulin-like receptor
  • said capture oligonucleotides further comprise a plurality of oligonucleotide subsets targeting blood group-determining polypeptide encoding nucleic acids ("blood group determining oligo subsets”)
  • said method further comprises the step of assigning to the sample, based on the hybridization pattern, a blood group phenotype; wherein each of said blood group determining oligo subsets comprises a set of overlapping oligonucleotides that cover every single nucleotide position in the mRNA sequences coding for the blood group-determining polypeptides from 5' to 3' and are sequentially shifted by 1-5 nucleotides from the 5' end of the preceding overlapping oligonucleotide.
  • said blood group determining polypeptides are selected from the group consisting ABO (ABO), Chido Rodgers (CH/RG), Colton (CO), Cromer (CROM), Diego (DI) (band 3), Dombrock (DO), Duffy (DARC), Gerbich (Ge), Gill (GIL), Globoside and Pk, H (H), I (I), Indian (IN), John Milton Hagen (JMH), Kell(KEL) and Kx(XK), Kidd (JK), Knops (KN), Landsteiner- Wiener (LW), Lewis (LE), Lutheran (LU), MNS (MNS, Glycophorins A, B and E), Ok (OK), Raph (RAPH), Rh(RH) and Rh-gp (RHAG), Scianna (SC), T/Tn, Xg (XG), and Yt (YT).
  • ABO ABO
  • CH/RG Chido Rodgers
  • CO Cromer
  • CROM Diego
  • DI band 3
  • the method further comprises the step of deriving from the HLA tissue type assigned in step (d) donor/recipient transplant compatibility.
  • the classical HLA oligo subsets comprise the sequences set forth in Table I or the normal (indicated by "HPN"), extended (indicated by "HPE") and truncated “indicated by HPT”) sequences set forth in Table X.
  • the non-classical HLA oligo subsets comprise the sequences set forth in Table II.
  • the accessory molecule oligo subsets comprise the sequences set forth in Table III.
  • the KIR oligo subsets comprise the sequences set forth in Table IV.
  • the blood group determining oligo subsets comprise the sequences set forth in Table V.
  • each oligonucleotide in each set of overlapping oligonucleotides, is sequentially shifted by 1 nucleotide from the 5' end of the preceding overlapping oligonucleotide. In another of the above embodiments, in each set of overlapping oligonucleotides, each oligonucleotide is sequentially shifted by 2 nucleotides from the 5' end of the preceding overlapping oligonucleotide.
  • each oligonucleotide in each set of overlapping oligonucleotides, is sequentially shifted by 3 nucleotides from the 5' end of the preceding overlapping oligonucleotide. In still other of the above embodiments, in each set of overlapping oligonucleotides, each oligonucleotide is sequentially shifted by 4 nucleotides from the 5' end of the preceding overlapping oligonucleotide.
  • each oligonucleotide in each set of overlapping oligonucleotides, is sequentially shifted by 5 nucleotides from the 5' end of the preceding overlapping oligonucleotide.
  • said sample for HLA tissue tying is obtained from a human subject.
  • the method for HLA tissue typing comprises the step of diagnosing or predicting the likelihood of an HLA-linked genetic defect, disease, inadequate or undesirable response to a vaccine, biologic treatment (recombinant protein, biosimilar or equivalent), or infectious organism, or condition in said subject, wherein the step is based on one or more assigned tissue types or phenotypes selected from the group consisting of: a classical HLA tissue type, a non-classical HLA tissue type, an accessory molecule phenotype, a KIR polypeptide phenotype, and a blood group phenotype.
  • the method for HLA tissue typing further comprises the step of determining the likely response of said subject to a particular treatment regimen selected from the group consisting of: bone marrow transplantation, immunosuppressive regimen, antiviral drug regimen, antiviral drug resistance, antiretroviral drug regimen, and autoimmunity drug regimen, wherein the step is based on one or more assigned phenotypes selected from the group consisting of: a classical HLA tissue type, a non-classical HLA tissue type, an accessory molecule phenotype, a KIR polypeptide phenotype, and a blood group phenotype.
  • the method for HLA tissue typing further comprises the step of determining whether the subject is likely to develop antiretroviral drug resistance or cancer drug regimen resistance, wherein the step is based on one or more assigned phenotypes selected from the group consisting of: a classical HLA tissue type, a non-classical HLA tissue type, an accessory molecule phenotype, a KIR polypeptide phenotype, and a blood group phenotype.
  • said capture oligonucleotides further comprise at least one set of negative control oligonucleotides.
  • said at least one set of negative control nucleotides comprises two or more of the nucleic acid sequences set forth in at least one of Tables VI-X .
  • a microarray comprising a substrate having disposed thereon capture oligonucleotides comprising the nucleic acid sequences set forth in Table I is provided.
  • a microarray comprising a substrate having disposed thereon capture oligonucleotides comprising the nucleic acid sequences set forth in Table I further has disposed thereon capture oligonucleotides comprising the nucleic acid sequences set forth in Table II.
  • a microarray comprising a substrate having disposed thereon capture oligonucleotides comprising the nucleic acid sequences set forth in Table X is provided.
  • a microarray comprising a substrate having disposed thereon capture oligonucleotides comprising the nucleic acid sequences set forth in Table X and denoted by "HPN,” "HPE” or “HPT” is provided.
  • a microarray comprising a substrate having disposed thereon capture oligonucleotides comprising the nucleic acid sequences set forth in Table II is provided.
  • the microarray having disposed thereon capture oligonucleotides comprising the nucleic acid sequences set forth in Table I and/or Table II further has disposed thereon capture oligonucleotides comprising the nucleic acid sequences set forth in one or more of Tables III-X.
  • Fig. 1 is a flow chart of HLA typing using an oligonucleotide microarray.
  • the whole process can be organized into three major steps.
  • the first step involves oligonucleotide "probe" design and microarray experiment.
  • the second step covers probe signal (i.e., fluorescent cRNA) preprocessing.
  • the third step covers the signal analysis programs for identification of sample HLA profiles.
  • Fig. 2 shows a set of oligonucleotides (PI to P 13) covering A*02:01 :01 :01 sequence and their corresponding negative control oligonucleotides (PIN to P13N) generated based on the consensus sequence of HLA class I sequence group.
  • the oligonucleotide sequences shown in Figure 2 have, from top to bottom, SEQ ID NOs: 76660-76686.
  • Fig. 3 shows a set of tiling capture oligonucleotides (P I to PI 3) covering the A*02:01 :01 :01 sequence.
  • the oligonucleotide sequences shown in Figure 3 have, from top to bottom, SEQ ID NOs: 76687-76700.
  • Fig. 4. is a graph demonstrating the relationship between target melting temperature and the capture oligonucleotide melting temperature range.
  • Fig. 5 contains pie graphs demonstrating the portioning of capture oligonucleotides with regard to (A) typing targets and (B) negative control oligonucleotides.
  • Fig. 6 shows histograms of capture oligonucleotide distribution according to (A) capture oligonucleotide length and (B) melting temperature.
  • Fig. 7 is a table displaying the maximum, minimum, mean, median, and geometric mean of the probe signals at position 31 of alignment of all HLA-A, B, C alleles.
  • Fig. 8 is flow diagram showing the approach for combining gap penalty method and pair-wise comparison method for identification of sample HLA alleles.
  • Fig. 9 contains graphs showing A*02010101 and A*03010101 probe signals of (A) sample D and (B) sample E.
  • the thick, darker lines plot probe signals at each alignment position and the thinner, lighter-colored lines plots thresholds (cutoffs) at each position.
  • the threshold used here is the 10% of the maximum signal at each position.
  • Fig. 10 shows an example of applying a pairwise comparison method on array E1A1A1 for predicting of HLA-A alleles.
  • A The input page of pairwise comparison method. A list of representative HLA-A alleles are input in the text box and array E1S 1A1 is chosen.
  • B Pairwise comparison is performed between each pair of the input alleles. The times of winning of one allele over the other are summarized in a table.
  • C The detailed signal comparison table of A*02010101 against A*03010101.
  • Each allele is indicated in the first column (on left); the starting nucleic acid position is shown in the second column; the oligonucleotide sequences are given in the third column, labeled "Probe", and have, numbered from top to bottom, SEQ ID NOs: 76701-76735; the melting temperature (in degrees Celsius) is shown in the fourth column; HLA coverage is shown in the fifth column and the numbers represent the number of different alleles; the sixth column shows the array signal for each oligo; and the seventh column shows the voting results - "similar” means there was no winner and if there was a winner, the winning allele is identified.
  • the polymorphic polypeptides are HLA polypeptides.
  • the invention provides an array comprising capture oligonucleotides that target nucleic acids encoding classical HLA polypeptides.
  • the array comprises capture oligonucleotides that target nucleic acids encoding non-classical HLA polypeptides.
  • the array comprises capture oligonucleotides that target nucleic acids encoding all known classical and non-classical HLA polypeptides, and in certain embodiments, the array additionally or alternatively comprises capture oligonucleotides that target nucleic acids encoding accessory molecules important in HLA-linked peptide presentation and/or antigen processing, killer- cell immunoglobulin-like receptor (KIR) polypeptides, and/or blood group determining polypeptides. All of these different targets (e.g., classical and non-classical HLA polypeptides accessory molecules, KIR polypeptides and blood group determining polypeptides) are referred to collectively herein as "array targets.”
  • a key feature of the methods and oligonucleotide arrays provided herein is that they provide an efficient and accurate method for determining the HLA tissue type of a sample using a single assay (e.g., array chip). This feature provides a dramatic improvement over currently available "traditional" technologies, which are inefficient because they typically require multiple assays and follow up nucleic acid sequencing in order to determine the complete HLA tissue type of a sample.
  • the arrays comprise sets of capture oligonucleotides that provide a highly dense coverage of the entire polypeptide-encoding nucleic acid sequence of each array target using an approach referred to herein as "walking.”
  • each oligonucleotide in a capture oligonucleotide set i.e. a set targeting one specific array target
  • sequential shifting by 1 nucleotide is also referred to as “step 1”
  • sequential shifting by 2 nucleotides is also referred to as "step 2”, and so on.
  • a first oligonucleotide has the sequence, ATGGCCGTCATGGCGCCCCGAAC (SEQ ID NO: 1)
  • the next oligonucleotide in the set if it is sequentially shifted by 1 nucleotide (i.e., a "step 1 shift") will have the sequence TGGCCGTCATGGCGCCCCGAAC (SEQ ID NO: 2)
  • that oligonucleotide is sequentially shifted by 2 nucleotides, rather than 1, it can have the sequence GGCCGTCATGGCGCCCCGAAC (SEQ ID NO: 3), and so on.
  • This approach produces a set of highly overlapping capture oligonucleotides, thereby providing dense coverage of each target sequence, which is important for high resolution typing of highly polymorphic genes such as HLA.
  • the arrays comprise capture oligonucleotides that target accessory molecules important in peptide loading on HLA molecules and/or antigen processing, and/or killer-cell immunoglobulin-like receptor (KIR) polypeptides, and/or blood group determining polypeptides.
  • KIR killer-cell immunoglobulin-like receptor
  • the array comprises capture oligonucleotides that target classical HLA molecules and/or non-classical HLA molecules (such arrays being referred to herein in general as "HLA oligonucleotide arrays"), and, in addition, comprises capture oligonucleotides that target accessory molecules important in peptide loading on HLA molecules and/or antigen processing, and/or KIR polypeptides, and/or blood group determining polypeptides.
  • HLA oligonucleotide array provides an efficient tool for obtaining a detailed analysis of a patient sample relevant for a wide variety of immunologic applications.
  • the arrays and methods of their use described herein are useful for diagnosing or predicting the likelihood of an HLA-linked genetic defect, disease, inadequate or undesirable response to a vaccine, biologic treatment (recombinant protein, biosimilar or equivalent), or infectious organism or condition in said subject, wherein the step is based on one or more assigned tissue types or phenotypes selected from the group consisting of: a classical HLA tissue type, a non-classical HLA tissue type, an accessory molecule phenotype, a KIR polypeptide phenotype, and a blood group phenotype.
  • the immunologic application of the arrays provided herein includes the ability to derive donor/recipient compatibility for bone marrow/tissue/organ transplant. Definitions
  • HLA polypeptide refers to an amino acid sequence encoded by a human leukocyte antigen (“HLA”) allele.
  • HLA haplotype refers to a combination of specific alleles from different HLA loci expressed as a combination of HLA genes characteristic for a given individual.
  • the term "all known" in the context of classical and/or non- classical HLA molecules means all of the HLA alleles for which the nucleic acid sequences are publically available at the time of assigning an HLA haplotype to a sample using the method claimed herein.
  • GenBank® National Institutes of Health (NIH) genetic sequence database
  • tissue typing refers to determining which of a number of isoforms and alleles of one or more families of polymorphic protein molecules are expressed in a cell (e.g., tissue).
  • families include, for example, classical HLA polypeptides, non-classical HLA polypeptides, KIR polypeptides, accessory molecule polypeptides, and blood group determining polypeptides.
  • HLA typing and "HLA tissue typing” are used interchangeably herein, and refer to the process that identifies the specific allele expressed for each gene at one or more of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP (classical HLA) gene loci in a sample (e.g., cells such as white blood cells, or cells derived from tissues).
  • a sample e.g., cells such as white blood cells, or cells derived from tissues.
  • the process described by these terms can also, but does not necessarily, include identifying the specific allele expressed for each gene at one or more of the HLA-E, HLA-F, HLA-G, DM, DO, and MIC (non-classical HLA) gene loci.
  • nucleic acid hybridization refers to the pairing of complementary strands of nucleic acids.
  • the mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of nucleic acids.
  • nucleobases complementary nucleoside or nucleotide bases
  • adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
  • Hybridization can occur under varying circumstances.
  • Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions.
  • Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters.
  • Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under "low stringency" conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid). See Molecular Biology of the Cell, Alberts et al, 3rd ed., New York and London: Garland Publ, 1994, Ch. 7.
  • hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over an extended portion of their length.
  • high stringency conditions include: hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, followed by washing in O.
  • lx SSC/0.1% SDS (where lx SSC is 0.15 M NaCl, 0.15 M Na citrate) at 68°C or for oligonucleotide (oligo) inhibitors washing in 6xSSC/0.5% sodium pyrophosphate at about 37°C (for 14 nucleotide- long oligos), at about 48°C (for about 17 nucleotide-long oligos), at about 55°C (for 20 nucleotide-long oligos), and at about 60°C (for 23 nucleotide-long oligos).
  • Conditions of intermediate or moderate stringency such as, for example, an aqueous solution of 2xSSC at 65°C; alternatively, for example, hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C followed by washing in 0.2 x SSC/0.1% SDS at 42°C
  • low stringency such as, for example, an aqueous solution of 2xSSC at 55°C
  • Specific temperature and salt conditions for any given stringency hybridization reaction depend on the concentration of the target DNA or RNA molecule and length and base composition of the probe, and are normally determined empirically in preliminary experiments, which are routine (see Southern, J.
  • standard hybridization conditions refers to hybridization conditions that allow hybridization of two nucleotide molecules having at least 50% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of only sequences having at least 75% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.
  • under hybridization conditions means conditions under conditions that facilitate specific hybridization of a subset of capture oligonucleotides to complementary sequences present in the cDNA or cRNA.
  • hybridizing specifically to and “specific hybridization” and “selectively hybridize to,” as used herein refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under at least moderately stringent conditions, and preferably, highly stringent conditions, as discussed above.
  • the term "complementary sequence,” when referring to a nucleic acid sequence, refers to the nucleic acid base sequence that can form a double-stranded structure (duplex) by matching bases to bases in a reference sequence.
  • the complementary sequence to the reference sequence G-T-A-C is C-A-T-G (SEQ ID NOs: 76736 and 76737, respectively).
  • a complementary sequence can have mismatches at certain nucleic acid residues with the reference sequence.
  • the "exact complement" of a reference nucleic acid sequence refers to a complementary sequence that contains no base mismatches with the reference sequence.
  • hybridization pattern refers to the raw data of a microarray assay, wherein, for example, the detectably labeled nucleic acid sample (e.g., cDNA or cRNA), or detectably labeled detection probes bound to nucleic acids (cDNA or cRNA sample) hybridized to capture oligonucleotides on the array, are detected in a specific pattern of detectable signal, the specific pattern being determined by which nucleic acid targets are present in the tested sample.
  • Hybridization patterns of different samples can also be compared visually, as long as the samples are hybridized to microarray slides having capture oligonucleotides spotted on the slides in identical locations. Hybridization patterns can be determined using, e.g., pattern recognition algorithms.
  • classical HLA oligo subset refers to a collection of capture oligonucleotides, the nucleic acid sequences of which collectively represent the nucleic acid sequence encoding a classical HLA polypeptide (e.g., a nucleic acid sequence encoding a specific HLA-A allele (e.g., HLA-A*0101 or HLA-A*0201)).
  • non-classical HLA oligo subset refers to a collection of capture oligonucleotides, the nucleic acid sequences of which collectively represent the nucleic acid sequence encoding a non-classical HLA polypeptide (e.g., a nucleic acid sequence encoding a specific HLA-E allele (e.g., HLA-E* 0101 or HLA-E* 0103)).
  • accessory molecule oligo subset refers to a collection of capture oligonucleotides, the nucleic acid sequences of which collectively represent the nucleic acid sequence encoding a target accessory molecule polypeptide
  • KIR oligo subset refers to a collection of capture oligonucleotides, the nucleic acid sequences of which collectively represent the nucleic acid sequence encoding a target KIR polypeptide
  • blood group determining oligo subset refers to a collection of capture oligonucleotides, the nucleic acid sequences of which collectively represent the nucleic acid sequence encoding a target blood group determining polypeptide.
  • KIR polypeptide phenotype refers to a specific set of KIR alleles expressed by a sample (e.g., cell), as determined according to the methods described herein.
  • blood group phenotype refers to the specific blood group of a sample (e.g., cell), including ABO (ABO), Chido Rodgers (CH/RG), Colton (CO), Cromer (CROM), Diego (DI) (band 3), Dombrock (DO), Duffy (DARC), Gerbich (Ge), Gill (GIL), Globoside and Pk, H (H), I (I), Indian (IN), John Milton Hagen (JMH), Kell(KEL) and Kx(XK), Kidd (JK), Knops (KN), Landsteiner- Wiener (LW), Lewis (LE), Lutheran (LU), MNS (MNS) (Glycophorins A, B and E), Ok (OK), Raph (RAPH), Rh(RH)and Rh-gp (RHAG), Scianna (SC), T/Tn, Xg (XG), and Yt (YT), as determined according to the methods described herein.
  • ABO ABO
  • the term can also, but does not necessarily, encompass a blood group subtype, such as subtype A 1 or A2.
  • the term "deriving donor/recipient transplant compatibility" means determining whether a tissue (cell, organ, skin, etc.) from a potential donor is suitable for transplantation into a recipient.
  • a suitable donor will have an HLA tissue type that is compatible (i.e. the same or highly similar) to that of the transplant recipient.
  • Polypeptide and “protein” are used interchangeably and mean any peptide- linked chain of amino acids, regardless of length or post-translational modification.
  • allele refers to a specific version of a nucleotide sequence of a polymorphic gene.
  • nucleic acid or “oligonucleotide” refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form.
  • the term also encompasses nucleic-acid-like structures with synthetic backbones.
  • DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem.
  • PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211 ; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144: 189-197. Other synthetic backbones encompassed by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156).
  • nucleic acid is used interchangeably with cDNA, cRNA, mRNA, oligonucleotide, probe and amplification product.
  • capture oligonucleotide refers to a nucleic acid sequence that specifically hybridizes to a target nucleic acid sequence (e.g., cDNA or cRNA).
  • a capture oligonucleotide is intended to be hybridized to a solid support (e.g., microarray slide) for the detection of the presence of a particular target sequence (e.g., cDNA or cRNA).
  • sample may be used herein to refer not only to detected nucleic acids, but to the detectable nucleic acids in the form in which they are applied to the target.
  • probe detection probe
  • nucleic acid probe are defined to be a collection of one or more nucleic acid fragments whose hybridization to a sample can be detected.
  • the probe may be unlabeled or labeled as described below so that its binding to the target or sample can be detected.
  • the probe is produced from a source of nucleic acids from one or more particular (preselected) portions of the genome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products.
  • PCR polymerase chain reaction
  • the probes of the present invention are synthesized and have sequences corresponding to a source of nucleic acids.
  • probes of the present invention correspond to or are produced from nucleic acids found in the regions described herein.
  • the probe or genomic nucleic acid sample may be processed in some manner, e.g., by removal of repetitive nucleic acids or enrichment with unique nucleic acids.
  • Probe signal means the level of fluorescence measured (e.g., by optical scanner) from hybridized oligonucleotides on the array.
  • nucleic acid array refers to a plurality of nucleic acid molecules (capture oligonucleotides) immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides and the like) to which sample nucleic acids (e.g., cDNA or cRNA) are hybridized.
  • the nucleic acids may contain sequence from specific genes or clones, such as the capture oligonucleotides of the invention, as disclosed herein. Other capture oligonucleotides optionally contain, for instance, reference sequences.
  • the capture oligonucleotides of the arrays may be arranged on the solid surface at different densities.
  • the capture oligonucleotides densities will depend upon a number of factors, such as the nature of the label, if any, the solid support, and the like.
  • isolated nucleic acid molecule is either (1) a nucleic acid molecule that contains sequence not identical to that of any naturally occurring sequence, or (2), in the context of a nucleic acid molecule with a naturally-occurring sequence (e.g., a cDNA, cRNA or genomic DNA), a nucleic acid molecule free of at least one of the genes that flank the gene containing the nucleic acid molecule of interest in the genome of the organism in which the gene containing the nucleic acid molecule of interest naturally occurs.
  • a nucleic acid molecule with a naturally-occurring sequence e.g., a cDNA, cRNA or genomic DNA
  • the term also includes a separate molecule such as: a cDNA where the corresponding genomic DNA has introns and therefore a different sequence; a genomic fragment that lacks at least one of the flanking genes; a fragment of cDNA or genomic DNA produced by polymerase chain reaction (PCR) and that lacks at least one of the flanking genes; a restriction fragment that lacks at least one of the flanking genes; a nucleic acid molecule encoding a non-naturally occurring protein such as a fusion protein, mutein, or fragment of a given protein; and a nucleic acid which is a degenerate variant of a cDNA or a naturally occurring nucleic acid.
  • a separate molecule such as: a cDNA where the corresponding genomic DNA has introns and therefore a different sequence; a genomic fragment that lacks at least one of the flanking genes; a fragment of cDNA or genomic DNA produced by polymerase chain reaction (PCR) and that lacks at least one of the flanking genes; a restriction
  • an isolated nucleic acid molecule does not mean a nucleic acid molecule present among hundreds to millions of other nucleic acid molecule molecules within, for example, cDNA or genomic DNA libraries or genomic DNA restriction digests in, for example, a restriction digest reaction mixture or an electrophoretic gel slice.
  • subject means any animal, including mammals and, in particular, humans.
  • HLA Human Leukocyte Antigen
  • HLA antigens are encoded by a series of closely linked genes located at the position p21 on chromosome 6. Genes of the HLA region span approximately 4 million bases of DNA, and are clustered into three distinct regions designated class I, class II and class III. Genes within the class I and class II regions share structural and functional properties and are considered to be part of the immunoglobulin gene super family. Although distinct in sequence and structure, both class I and class II genes encode proteins that are critical in controlling T-cell recognition and determining histocompatibility in marrow transplantation (Rammensee, Curr. Opin. Immunol. 7:85-96 (1995)).
  • the HLA-A, -B and -C loci show a striking degree of sequence and structural homology with one another and genes at all three loci are highly polymorphic (Bodmer et al, Tissue Antigens 49:297-321 (1997)).
  • Class II genes are divided into five families, designated DR, DQ, DO, DM and DP, based on their degree of sequence homology and their location within the HLA-D region.
  • the HLA class II region is comprised of nine distinct genes: DRA, DRBl, DRB3, DRB4, DRB5, DQA, DQB, DPA and DPB.
  • class II DR, DQ and DP genes show a striking degree of polymorphism, with more than 805 alleles thus far defined at the DRBl locus (Marsh SG, Hum Immunol, (2010)).
  • the microarrays described herein detect expression of non-classical HLA molecules.
  • non-classical HLA molecules include without limitation, CDla-c, CD Id, HLA-E, HLA-G, HLA-H, HLA- J, HLA-K, HLA-L, DM, DOa, DOP, ULBP, EPCR, MRl, FcRn, HFE, ZAG, and MIC.
  • the non-classical HLA molecules detected by the arrays provided herein include at least HLA-E, HLA-F, HLA-G, DM, DO and MIC, although a greater or fewer number of non-classical HLA targets can be included on the arrays.
  • antigen-presenting cells engulf the pathogen through a process called phagocytosis.
  • HLA molecules specifically MHC class II
  • the peptides are then displayed by the APCs to T cells, which then produce a variety of effects to eliminate the pathogen.
  • proteins both native and foreign, such as the proteins of viruses
  • HLA antigens specifically MHC class I
  • Infected cells can be recognized and destroyed by components of the immune system (specifically CD8+ T cells).
  • Peptides such as, e.g., infection or disease-related peptides fit into the binding clefts of HLA molecules, and, in these configurations, peptides are presented to T cells.
  • the T cells are restricted by the HLA molecules when certain peptides are within the binding cleft.
  • Each HLA molecule is limited in the number of peptides (e.g. disease or infection related peptides) that it can bind.
  • HLA tissue type which increases the peptide binding repertoire, is important for how an individual's immune system can respond to infection.
  • the ability to precisely characterize an individual's HLA tissue type can therefore facilitate understanding of how an individual's immune system can respond to a particular infection.
  • a large number of studies have reported correlation with specific HLA profiles and susceptibility to or severity of disease.
  • Transplant rejection occurs when a transplanted organ or tissue is not accepted by the body of the transplant recipient, typically because the transplanted organ or tissue is recognized by the recipient's immune system as non-self. Any cell displaying an HLA type not expressed by the recipient is recognized as "non-self, resulting in the rejection of the tissue bearing those cells.
  • tools and methods for carrying out HLA tissue typing with precision and efficiency in order to quickly determine whether a potential donor has an HLA tissue type that is compatible with that of a recipient.
  • HLA types are inherited, and some of them are connected with autoimmune disorders and other diseases. People with certain HLA tissue types are more likely to develop certain autoimmune diseases, such as Type I Diabetes, Rheumatoid arthritis, Ankylosing spondylitis, Celiac Disease, SLE (Systemic Lupus Erythematosus), Myasthenia Gravis, inclusion body myositis and Sj5gren's syndrome.
  • autoimmune diseases such as Type I Diabetes, Rheumatoid arthritis, Ankylosing spondylitis, Celiac Disease, SLE (Systemic Lupus Erythematosus), Myasthenia Gravis, inclusion body myositis and Sj5gren's syndrome.
  • Celiac disease HLA tissue typing is the only effective means of discriminating between 1 st degree relatives who are at risk from those who are not, prior to the appearance of sometimes irreversible symptoms such as allergies and secondary autoimmune disease.
  • DQ2 typing For HLA typing to lead to some improvement and acceleration in the diagnosis of Celiac Disease and Type 1 diabetes, DQ2 typing is necessary. However, for DQ2 typing to be useful it requires either high resolution Bl*typing (resolving *0201 from *0202), DQAl *typing, or DR serotyping.
  • the arrays provided herein, which provide high resolution typing of classical HLA polypeptides, and preferably, all known classical HLA polypeptides, are useful for diagnosing such disorders. Role of HLA in cancer
  • HLA mediated diseases are directly involved in the promotion of cancer.
  • gluten sensitive enteropathy is associated with increased prevalence of enteropathy-associated T-cell lymphoma, and DR3-DQ2 homozygotes are within the highest risk group with close to 80% of gluten sensitive EATL cases.
  • the arrays provided herein are useful in diagnosing, e.g., an individual's risk of developing cancer based on the assigned HLA tissue type of the individual (see, e.g., Luigi De Petris, et al. Medical Oncology, Volume 21, Number 1, 49-52, DOI: 10.1385/MO:21 : 1 :49; Lin P, et al. Cancer Epidemiol Biomarkers Prev. 2001 Oct; 10(10): 1037-45; Michallet et al, Leukemia. 10: 1725- 31, (2010)).
  • the microarrays described herein can detect expression of accessory molecules important in HLA-linked peptide presentation and/or antigen processing, including, for example, and without limitation, LMP2, LMP7, LMP10, tripeptidyl peptidase II (TPPII), bleomycin hydrolase (BLMH), leucine aminopeptidase 3 (LAP3), transporter associated with antigen processing (TAP) 1, TAP2, p2-microglobulin, TAP binding protein (tapasin), calnexin (CANX), calreticulin (CALR), protein disulfide isomerase family A member 2 (PDIA2), protein disulfide isomerase family A member 3 (PDIA3), ERp57, endoplasmic reticulum aminopeptidase (ERAP) 1, ERAP2, proteasome (prosome macropain) subunit althap (PSMA) type I (PSMA1), PSMA2, PSMA3, PSMA4, PSMA5, PSMA6, PSMA7,
  • the arrays provided herein can simultaneously determine HLA tissue type and an accessory molecule phenotype (expression of one or more accessory molecules important in peptide loading and/or antigen processing) in a single assay (e.g. on a single microarray slide).
  • the array specific for detecting expression of such accessory molecules can be carried out separately (e.g., on a separate microarray slide and/or in a separate assay).
  • arrays comprising both HLA oligonucleotides (classical and/or non-classical HLA oligonucleotides) and accessory molecule oligonucleotides targeting, e.g., the accessory molecules described above, are useful for determining donor/recipient transplant compatibility.
  • such combined HLA/accessory molecule arrays are useful for diagnosing or predicting the likelihood of an HLA-linked genetic defect, disease, inadequate or undesirable response to a vaccine, biologic treatment (recombinant protein, biosimilar or equivalent), or infectious organism, or condition in a subject, wherein the step is based on one or more assigned HLA tissue types (including classical and/or non-classical HLA alleles) and an accessory molecule phenotype.
  • such combined HLA/accessory molecule arrays are useful for determining the likely response of a subject to a particular treatment regimen selected from the group consisting of: bone marrow transplantation, immunosuppressive regimen, antiviral drug regimen, antiviral drug resistance, antiretroviral drug regimen, and autoimmunity drug regimen, wherein the step is based on one or more assigned HLA tissue types (including classical and/or non-classical HLA alleles) and an accessory molecule phenotype.
  • such combined HLA/accessory molecule arrays are useful for determining whether the subject is likely to develop antiretroviral drug resistance or cancer drug regimen resistance, wherein the step is based on one or more assigned HLA tissue types (including classical and/or non-classical HLA alleles) and an accessory molecule phenotype.
  • KIR Killer-cell Immunoglobulin-like Receptor
  • KIR molecules Killer-cell immunoglobulin-like receptors
  • NK natural killer cells.
  • KIR molecules regulate the killing function of NK cells by interacting with MHC class I molecules, which are expressed on all cell types. This interaction allows them to detect virally infected cells or tumor cells that have a characteristic low level of Class I MHC on their surface.
  • MHC class I molecules are expressed on all cell types. This interaction allows them to detect virally infected cells or tumor cells that have a characteristic low level of Class I MHC on their surface.
  • Most KIR molecules are inhibitory, meaning that their recognition of MHC suppresses the cytotoxic activity of the NK cell. Only a limited number of KIRs have the ability to activate cells.
  • KIR genes are highly polymorphic, so that different individuals possess different arrays/repertoires of KIR genes.
  • the polymorphic KIR genes are found in a cluster on chromosome 19ql3.4 within the 1 Mb leukocyte receptor complex (LRC).
  • LRC leukocyte receptor complex
  • the gene content of the KIR gene cluster varies among haplotypes, although several "framework" genes are found in all haplotypes (KIR3DL3, KIR3DP1, KIR3DL4, KIR3DL2).
  • the KIR proteins are classified by the number of extracellular immunoglobulin domains (2D or 3D) and by whether they have a long (L) or short (S) cytoplasmic domain.
  • KIR proteins with the long cytoplasmic domain transduce inhibitory signals upon ligand binding via an immune tyrosine-based inhibitory motif (ITIM), while KIR proteins with the short cytoplasmic domain lack the ITIM motif and instead associate with the TYRO protein tyrosine kinase binding protein to transduce activating signals.
  • ITIM immune tyrosine-based inhibitory motif
  • the ligands for several KIR proteins are subsets of HLA class I molecules; thus, KIR proteins are thought to play an important role in regulation of the immune response.
  • KIR involvement in tissue transplant has been reported. Specifically, it has been reported that certain combinations of HLA and KIR haplotypes may affect outcome in T-cell depleted haematopoietic stem cell transplantation (HSCT) (Chen, C. et al. Bone Marrow Transplantation (2006) 38, 437 ⁇ 144). Thus, the determination of the KIR phenotype of an individual can be useful for determining donor/recipient transplant compatibility.
  • KIR molecules the expression of which can be determined by the assays and method provided herein, include without limitation, KIR 2DL1, 2DL2, 2DL3, 3DL1, 3DL2, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, and 3DS1.
  • the present arrays provide the ability to simultaneously determine both HLA tissue type and KIR haplotype of a sample on a single array, which provides a highly efficient and informative assay for determining donor/recipient compatibility, in addition to other diagnostic uses.
  • such combined HLA/KIR polypeptide arrays are useful for diagnosing or predicting the likelihood of an HLA-linked genetic defect, disease, inadequate or undesirable response to a vaccine, biologic treatment (recombinant protein, biosimilar or equivalent), or infectious organism, or condition in a subject, wherein the step is based on one or more assigned HLA tissue types (including classical and/or non-classical HLA alleles) and a KIR phenotype.
  • such combined HLA/KIR polypeptide arrays are useful for determining the likely response of a subject to a particular treatment regimen selected from the group consisting of: bone marrow transplantation, immunosuppressive regimen, antiviral drug regimen, antiviral drug resistance, antiretroviral drug regimen, and autoimmunity drug regimen, wherein the step is based on one or more assigned HLA tissue types (including classical and/or non-classical HLA alleles) and a KIR phenotype.
  • such combined HLA/KIR polypeptide arrays are useful for determining whether the subject is likely to develop antiretro viral drug resistance or cancer drug regimen resistance, wherein the step is based on one or more assigned HLA tissue types (including classical and/or non-classical HLA alleles) and KIR phenotype.
  • Distinct molecules called agglutinogens are attached to the surface of red blood cells.
  • agglutinogens There are two different types of agglutinogens, type "A” and type "B". Each type has different properties.
  • the ABO blood type classification system uses the presence or absence of these molecules to categorize blood into four types: A, B, AB, and O.
  • A, B, AB, and O In each individual, two alleles encoding the enzymes responsible for determining blood group antigens are inherited, one from each parent.
  • the possible combinations of alleles produce blood types in the following way: AA, AB, AO, BA BB, BO, OA, OB, OO.
  • the A and B antigen molecules on the surface of red blood cells are produced by two different enzymes. These two enzymes are encoded by different versions, or alleles, of the same gene: A and B.
  • the A and B alleles code for enzymes that produce the type A and B antigens respectively.
  • the A allele encodes a glycosyltransferase that bonds a-N-acetylgalactosamine to the D-galactose end of the H antigen, producing the A antigen.
  • the B allele encodes a glycosyltransferase that joins a-D-galactose bonded to the D-galactose end of the H antigen, creating the B antigen.
  • a third version of this gene contains a deletion of a single nucleotide (guanine at position 261 in exon 6), which results in loss of enzymatic activity of the encoded protein, thereby leading to failure to modify the H antigen.
  • Another level of specificity is added to blood type by examining the presence or absence of the Rh protein.
  • Each blood type is either positive "+” (has the Rh protein) or negative "-" (no Rh protein).
  • a + a person whose blood type is "A positive” (A +), has both type A and Rh proteins on the surface of their red blood cells.
  • Determining the blood type of a sample, in addition to the HLA tissue type can be useful, e.g., for the determination of donor/recipient compatibility for e.g., tissue transplant.
  • the arrays provided herein provide an efficient method for simultaneously determining both HLA tissue type and blood group in a single assay. In the transplant field, in particular, it is critical that donor/recipient compatibility results be obtained as quickly and cost-efficiently as possible, and the efficiency of the arrays provided herein address that need.
  • the A, B and O blood types also contain subgroups. For example, there are six common alleles in white individuals of the ABO gene that produce one's blood type, including A101 (Al), A201 (A2), B101 (Bl), O01 (01), O02 (Olv), and O03 (02).
  • the different blood groups, and in some cases specific subgroups, can be associated with different diseases, and thus, determining the specific allele of a cell can be important for determining disease susceptibility.
  • O group compared to non-0 group (A, AB, and B) individuals have a 14% reduced risk of squamous cell carcinoma and 4% reduced risk of basal cell carcinoma.
  • O group is also associated with a reduced risk of pancreatic cancer.
  • the B antigen is linked with increased risk of ovarian cancer, and gastric cancer has been reported to be more common in blood group A and least in group O.
  • Non-limiting examples of blood group determining molecules that can be determined according to the arrays and methods provided herein include ABO (ABO), Chido Rodgers (CH/RG), Colton (CO), Cromer (CROM), Diego (DI) (band 3), Dombrock (DO), Duffy (DARC), Gerbich (Ge), Gill (GIL), Globoside and Pk, H (H), I (I), Indian (IN), John Milton Hagen (JMH), Kell(KEL) and Kx(XK), Kidd (JK), Knops (KN), Landsteiner- Wiener (LW), Lewis (LE), Lutheran (LU), MNS (MNS, Glycophorins A, B and E), Ok (OK), Raph (RAPH), Rh(RH)and Rh-gp (RHAG), Scianna (SC), T/Tn, Xg (XG), and Yt (YT).
  • ABO ABO
  • Chido Rodgers CH/RG
  • Colton CO
  • Cromer C
  • the characterization of blood group determining molecules by the arrays and methods provided herein are useful, e.g., in the context of blood transfusions.
  • the compatibility of the blood donor and recipient Prior to blood transfusion, the compatibility of the blood donor and recipient is dependent upon the blood group factors mentioned above. Compatibility is a clinical necessity due to the fatal consequences of blood agglutination which can result from incompatibility.
  • compatible donors can be selected.
  • the solid supports e.g., microarray slides, used in the present invention may be biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc.
  • the solid support is preferably flat but may take on alternative surface configurations.
  • the solid support may contain raised or depressed regions on which synthesis takes place.
  • the solid support will be chosen to provide appropriate light-absorbing characteristics.
  • the support may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, S1O 2 , S1N 4 , modified silicon, or any one of a variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene, polycarbonate, or combinations thereof.
  • suitable solid support materials will be readily apparent to those of skill in the art.
  • the surface of the solid support will contain reactive groups, which could be carboxyl, amino, hydroxyl, thiol, or the like. More preferably, the surface will be optically transparent and will have surface Si-OH functionalities, such as are found on silica surfaces.
  • Attached to the solid support is an optional spacer or linking group.
  • the spacer molecules are preferably of sufficient length to permit the capture oligonucleotides in the completed array to interact freely with molecules exposed to the array.
  • the spacer molecules when present, are typically 6-50 atoms long to provide sufficient exposure for the attached probes.
  • the spacer will typically be comprised of a surface attaching portion and a longer chain portion.
  • the surface attaching portion is that part of the linking group or spacer which is directly attached to the solid support. This portion can be attached to the solid support via carbon-carbon bonds using, for example, supports having (poly)trifluorochloroethylene surfaces, or preferably, by siloxane bonds (using, for example, glass or silicon oxide as the solid support).
  • Siloxane bonds with the surface of the support are formed in one embodiment via reactions of surface attaching portions bearing trichlorosilyl or trialkoxysilyl groups.
  • the surface attaching groups will also have a site for attachment of the longer chain portion.
  • groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl.
  • Preferred surface attaching portions include aminoalkylsilanes and hydroxyalkylsilanes.
  • the surface attaching portion of the linking group is either aminopropyltriethoxysilane or aminopropyltrimethoxysilane.
  • the longer chain portion can be any of a variety of molecules which are inert to the subsequent conditions necessary for attaching the oligonucleotide probes, or for hybridization of a sample to the oligonucleotide array. These longer chain portions will typically be ethylene glycol oligomers containing 2-14 monomer units, diamines, diacids, amino acids, peptides, or combinations thereof. In some embodiments, the longer chain portion is a polynucleotide (e.g., a 15-mer of poly dT).
  • the linking group will typically have a protecting group, attached to a functional group (i.e., hydroxyl, amino or carboxylic acid) on the distal or terminal end of the chain portion (opposite the solid support). After deprotection and coupling, the distal end is covalently bound to a capture oligonucleotide (e.g., an HLA-A capture oligonucleotide).
  • a functional group i.e., hydroxyl, amino or carboxylic acid
  • Presynthesized capture oligonucleotides can be delivered to a solid support by high-speed robotics, and then immobilized on the surface.
  • the resolution of the resulting oligonucleotide array is determined by both the spatial resolution of the delivery systems and the physical space requirement of the delivered oligonucleotide solution volume.
  • the surface density of the immobilized capture oligonucleotides varies greatly with different solid surface and linkage chemistries (Guo, et al, Nuc. Acids Res. 22:5456-5465 (1994); Fahy, et al, Nuc. Acids Res. 21 : 1819-1826 (1993); Wolf, et al, Nuc. Acids Res. 15:2911-2926 (1987); and Ghosh, et al, Nuc. Acids Res. 15:5353-5372 (1987)).
  • capture oligonucleotides are synthesized directly onto the solid support, nucleotide by nucleotide, through a series of coupling and deprotection steps.
  • Both conventional solid-phase oligonucleotide synthesis methods and light-directed combinatorial synthesis methods have been successfully applied in this in situ fabrication process (Fodor et al, supra (1991) and Gilham, Biochemistry 7:2809-2813 (1968)).
  • High reaction yields in both the coupling and the deprotection steps are critical for the success of in situ synthesis.
  • the preparation of in situ arrays can be automated and thereby increase the complexity of the array compared to the use of presynthesized oligonucleotides.
  • a capture oligonucleotide is immobilized onto a solid support through a single covalent bond.
  • Gilham Biochemistry, 7:2809-2813 (1968)
  • Suitable supports for covalent immobilization of DNA include glass, acrylamide gel, latex particles, controlled pore glass, dextran supports, polystyrene matrices and avidin-coated polystyrene beads and have been described (Guo, et al., Nuc. Acids Res. 22:5456-5465 (1994); Fahy, et al, Nuc. Acids Res.
  • Fodor, et al. demonstrated the use of photolithographic technology to synthesize high-density oligonucleotide arrays on silicon substrates.
  • crosslinkers are first made by exposing a photochemically-labile organosilane surface to UV light. The resulting pattern is then reacted with heterobifunctional crosslinking molecules. The oligonucleotide molecules are then bound to these crosslinkers to form a well-defined DNA pattern on the surface. Spatial resolution of 1 micron per DNA spot is feasible using this approach.
  • Three-dimensional immobilization matrices have been developed to increase capacity and are contemplated for use for the array provided herein.
  • Yershor, et al. (Genetics 93 :4913-4918 (1996)), for example, have produced DNA arrays by immobilizing oligonucleotides in acrylamide gel at a density of 20,000 to 30,000 different capture oligonucleotides per cm 2 , two orders of magnitude higher than the capacity of two- dimensional supports, with density increasing as technology advances.
  • the three- dimensional support permits high oligonucleotide loading and enhanced hybridization.
  • the application of this approach is limited.
  • Solid phase DNA synthesis can be accomplished with a number of different chemistries. Froehler et al. (Nuc. Acids Res. 14:5399-5407 (1986) and McBride and Caruthers (Tetrahedron Lett. 24:245-248 (1983) have demonstrated solid phase DNA synthesis chemistries utilizing H-phosphonate and phosphoramidites, which covalently attach an organic linker molecule to a surface and build the oligonucleotide off the terminus of the linker through successive coupling and deprotection steps.
  • Microfabricated ink-jet pumps similar to those used in certain ink-jet printers to deliver synthesis reagents onto the surface of a solid support can also be used.
  • the surface is scanned across a set of ink-jet pumps using a computer-controlled x-y translation stage.
  • DNA monomers are delivered to the defined area at rates of several hundred drops per second.
  • oligonucleotide arrays described herein can be prepared according to the above-described methods or according to any other suitable method known in the art.
  • oligonucleotide arrays can be prepared by:
  • the solid supports can be any of those described above which are conveniently derivatized with a vapor phase deposition of an aminoalkyltrialkoxysilane.
  • the aminoalkyltrialkoxysilanes useful in this aspect of the invention are any of those that can be utilized in the vapor phase at temperatures of from about ambient temperature to about 150 °C at pressures of from about 760 mmHg to about 0.1 mmHg.
  • the aminoalkyl portion of the silane will be aminopropyl, aminoethyl or aminomethyl.
  • the trialkoxysilane portion can be one in which the alkoxy groups are all the same (e.g., trimethoxysilane, triethoxysilane) or one in which the alkoxy groups are not all alike (e.g., dimethoxyethoxysilane).
  • the aminoalkyltrialkoxysilane will typically be selected from aminopropyltrimethoxysilane, aminopropyltriethoxysilane, aminopropyldiethoxy-methoxysilane, aminoethyltrimethoxysilane, and the like. More preferably, the aminoalkyltrialkoxysilane is aminopropyltrimethoxysilane.
  • a more uniform coating of amino groups on the solid support can be achieved by applying an aminoalkyltrialkoxysilane in the vapor phase, typically at reduced pressure. This can be accomplished by placing the solid support into a vacuum chamber, evacuating the chamber, and introducing the silane.
  • the vacuum chamber can be heated to facilitate silane vaporization and even coating of the solid support.
  • the pressure will typically be from about 5 to 35 mmHg and the vacuum chamber will be heated to a temperature of from about 60 to about 110 °C.
  • the resultant support can then be contacted with a suitable amount of a linking group to covalently attach the linking group to the aminoalkylsilane-derivatized solid support.
  • the aminoalkylsilane-derivatized solid support will first be treated with a reagent capable of facilitating linking group attachment to the derivatized support.
  • a reagent capable of facilitating linking group attachment to the derivatized support is useful in this aspect of the invention including diisocyanates, diisothiocyanates, dicarboxylic acids (and their activated esters), and the like. Particular preferred are diisothiocyanates (e.g., 1 ,4-phenylenediisothiocyanate).
  • linking group is attached to provide a spacing between the oligonucleotide probe and the support which is optimized for interactions between the probes and the sample.
  • a variety of linking groups can be used in this aspect of the invention. Preferred groups are those that provide a spacing similar to that provided by a 15-mer poly dT spacing group.
  • the linking group will have a reactive portion that is selected to be compatible with the amino group of the aminoalkylsilane-derivatized support, or with the functional group present on the reagent used to facilitate linking group attachment (e.g., the isothiocyanate portion of 1,4- phenylenediisothiocyanate).
  • the linking group will have a functional group that is reactive with an amino moiety (e.g., a carboxylic acid, anhydride, isothiocyanate, and the like) or a functional group that is reactive with an isocyanate, isothiocyanate or carboxylic acid moiety (e.g., an amino group, a hydroxy 1 group or the like).
  • an amino moiety e.g., a carboxylic acid, anhydride, isothiocyanate, and the like
  • an isocyanate, isothiocyanate or carboxylic acid moiety e.g., an amino group, a hydroxy 1 group or the like.
  • the support is derivatized first with aminopropyltrimethoxysilane, followed by attachment of 1,4-phenylenediisothiocyanate, followed by attachment of a 15-mer oligonucleotide, preferably a 15-mer of poly-dT.
  • the capture oligonucleotides can be any collection of nucleic acids or polymer.
  • the capture oligonucleotides are those that represent one or more of the groups selected from all known classical HLA polypeptides, all known non-classical HLA polypeptides, accessory molecules important in antigen processing and presentation, KIR polypeptides, and blood group determining polypeptides.
  • the capture oligonucleotides are typically 17 to 60 nucleotides in length, although shorter or longer sequences are also contemplated, so that each has a melting temperature (T m ) with respect to its exact complement of about 64 °C (e.g., about 64.0, 64.1, 64.2, 64.3, 64.4, 64.5 or 64.6 °C). In a specific embodiment, the preferred Tm is about 64.3.
  • T m melting temperature
  • Preferred capture oligonucleotides have the nucleic acid sequences set forth in Tables I-V, below.
  • the capture oligonucleotides can be prepared by any conventional methods known to those of skill in the art.
  • the oligonucleotides can be constructed on the array using the techniques described above (e.g., photolithography, flow channel, ink-jet spotting, and the like).
  • the oligonucleotides are constructed using conventional solution or solid phase chemistry, and then attached to the array's solid support component (e.g., slide).
  • Construction of the present arrays is preferably carried out in a manner that ensures that the capture oligonucleotides are present at a surface density of about 250 to about 450 angstrom 2 /molecule, preferably about 325 to about 375 angstrom 2 /molecule, or higher. Methods of measuring oligonucleotide density are well known to those of skill in the art. Hybridization
  • Hybridization of DNA to a solid support has similar thermodynamic behavior compared to hybridization of DNA in solution.
  • the stability of the double helix can be characterized by its melting temperature, which is strongly dependent upon oligonucleotide sequence and composition of the solvent (Wetmur, Crit. Rev. Biochem. & Mol. Bio. 26:227- 259 (1991)).
  • This strong-dependence of the duplex stability on oligonucleotide sequence especially for short oligonucleotides, makes it difficult to design adequately stringent conditions for hybridization with oligonucleotide arrays, which usually vary widely in base composition. Thus, a large number of false positive or negative signals may occur when hybridization is performed on complex oligonucleotide arrays.
  • TMAC tetramethylammonium chloride
  • thermodynamic stability of solid-phase hybridization is also affected by differences between the perfectly matched duplex versus the mismatched duplex, which constitutes the fundamental limitation to sequence-specific recognition in hybridization.
  • the binding of a capture oligonucleotide mismatched at a single base is compared with that of a perfectly matched capture oligonucleotide (i.e., its "exact complement"); the difference in duplex stability is used to identify the target sequence.
  • the differences in stability of a perfect match and a single-base mismatch are so small that discrimination between a perfect match and a single base mismatch cannot be achieved using common hybridization-washing procedures. Guo et al.
  • hybridization kinetics was found to be proportional to the concentration of immobilized DNA.
  • a mathematical model of hybridization on solid supports has been proposed that hypothesizes two different mechanisms by which nucleic acid targets can hybridize with immobilized oligonucleotide probes: direct hybridization from solution and hybridization by nucleic acid targets that adsorb nonspecifically on the surface and then diffuse to the capture oligonucleotides (Chan et al, Biophysical J. 69:2243-2255 (1995)).
  • the hybridization rate depends strongly on both the nucleic acid diffusion constant in solution and the nucleic acid adsorption/desorption constant on surface.
  • the nucleic acid sample (e.g., cDNA or cRNA) is directly labeled with a detectable marker.
  • cDNA or cRNA can be made from RNA obtained from a sample (e.g. blood or tissue) and fluorescent labels (e.g., dyes) can be incorporated into the cDNA or cRNA.
  • the nucleic acid samples themselves are not detectably labeled, but labeled detection probes are used that bind to target nucleic acids hybridized to capture oligonucleotides on the arrays.
  • nucleic acid targets including cDNA, cRNA and detection probes
  • hybridization systems can be fluorescently labeled either directly or indirectly.
  • the direct fluorescent label systems for nucleic acid molecules include derivatives of fluorescein and rhodamine dyes, which can be easily attached to the end of a nucleic acid strand.
  • Biotin is the most commonly used indirect fluorescent label. Biotin can be easily incorporated into nucleic acid molecules and detected using avidin or streptavidin by a covalently linked reporter group, such as alkaline phosphatase and horseradish peroxidase (Rees and Kurz, Nuc. Acids Res. 12:3435-3439 (1984)).
  • a covalently linked reporter group such as alkaline phosphatase and horseradish peroxidase (Rees and Kurz, Nuc. Acids Res. 12:3435-3439 (1984)).
  • alkaline phosphatase and horseradish peroxidase Rees and Kurz, Nuc. Acids Res. 12:3435-3439 (1984)
  • the fluorescence detection systems require that excess label be washed off; furthermore, after hybridization real-time monitoring of the hybridization process is not feasible.
  • surface-related detection methods have been developed. These methods are based on different optical phenomenon on the surface and can detect subtle changes such as the formation of nucleic acid duplexes on the surface, without interfering with the excess nucleic acid in solution.
  • Duplex electron transfer, optical wave-guide, surface plasmon resonance and resonant mirror are a few examples of currently developed surface-based detection methods (Wood, Microchem. J.
  • the present invention provides an array of capture oligonucleotides specific for nucleic acids encoding HLA polypeptides.
  • the array is a useful tool for performing HLA tissue typing on a cell or tissue sample to determine, for example, whether a particular donor is suitable for matching in bone marrow, tissue (e.g., skin or organ) transplantation.
  • the array will comprise a series of capture oligonucleotides which represent at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably 100% of all known classical HLA polypeptides.
  • the array will comprise a series of capture oligonucleotides which represent at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably 100% of all known non-classical HLA polypeptides.
  • the arrays will represent all known non- classical HLA polypeptides.
  • the arrays will represent all known classical HLA polypeptides and/or all known non-classical HLA polypeptides.
  • the capture oligonucleotides are provided on the array at known or preselected positions to facilitate analysis. Additionally, the capture oligonucleotides are generally covalently attached to the solid support using a linking group that is sufficient to provide optimum binding of a sample nucleic acid to the oligonucleotide array.
  • the above-described HLA oligonucleotide arrays (which can comprise capture oligonucleotides specific for either classical HLA polypeptides or non-classical HLA polypeptides or specific for both classical and nonclassical HLA polypeptides), further comprise negative control oligonucleotides.
  • the arrays provided herein comprise capture oligonucleotides that target nucleic acids encoding all known classical and non-classical HLA polypeptides, and, additionally or alternatively, comprise other capture oligonucleotides that target nucleic acids encoding other array targets, including but not limited to accessory molecules important in HLA-linked peptide presentation and/or processing, KIR polypeptides, and/or blood group determining polypeptides.
  • negative control capture oligonucleotides can be optionally be included in the array, or can be included on a separate array (e.g., separate slide).
  • capture oligonucleotides are designed to represent at least 80%, preferably at least 90% and more preferably at least 98% of the target polypeptide-encoding nucleic acid sequences (e.g., all classical and/or non-classical HLA polypeptides).
  • each oligonucleotide in a set of overlapping oligonucleotides is sequentially shifted by 1, 2, 3, 4, 5 or more nucleotides from the 5' end of the preceding overlapping oligonucleotide (i.e., each oligonucleotide contains a step 1, step 2, step 3, step 4, or step 5 shift from the preceding oligonucleotide in the set).
  • capture oligonucleotides In order for the melting temperature of the capture oligonucleotide sequences to be comparable, capture oligonucleotides should be designed with careful attention to size, base composition, and placement of mismatched position within the hybridization sequence. Within certain embodiments, capture oligonucleotides can range in length from about 5 nucleotides (nt) to about 80 nt, about 7 nt to about 75 nt, about 10 nt to about 70 nt, about 15 nt to about 65 nt, or, preferably, about 17 nt to about 60 nt in length.
  • nt nucleotides
  • the melting temperature (T m ) of the capture oligonucleotides can range from about 64.3 ⁇ 0.7°C. In a preferred embodiment, all capture oligonucleotides have a Tm of about 64.3°C.
  • the oligonucleotide array comprises the capture oligonucleotides having the nucleic acid sequences set forth in Table I, below.
  • the oligonucleotides in Table I are designed to collectively target transcripts (e.g., cDNA or cRNA) encoding target classical HLA molecules.
  • Table I provides an example of a complete set of capture oligonucleotides targeting classical HLA molecules.
  • Tables II-V and X, below, set forth exemplary sequences of a partial set of capture oligonucleotides directed to the indicated molecules.
  • One of skill in the art will understand how to design the complete set of capture oligonucleotides based on the teachings provided herein, e.g., in the Examples, below, and based on the complete set of oligonucleotides exemplified in Table I.
  • negative control oligonucleotides as set forth in Tables VI-IX, as well as the oligonucleotides designated by "H " in Table X, are provided as non-limiting examples of negative control oligonucleotides.
  • the skilled artisan will understand how to design a complete set of negative control oligonucleotides based on the teachings provided herein.
  • the oligonucleotide array comprises the capture oligonucleotides having the nucleic acid sequences set forth in Table II, below.
  • the oligonucleotides in Table II are examples of the oligonucleotides that can be designed to collectively target transcripts (e.g., cDNA or cRNA) encoding non-classical HLA molecules.
  • cDNA or cRNA collectively target transcripts
  • the oligonucleotide array comprises the capture oligonucleotides having the nucleic acid sequences set forth in Table III, below.
  • the oligonucleotides in Table III are examples of the oligonucleotides that can be designed to collectively target transcripts (e.g., cDNA or cRNA) encoding accessory molecules.
  • target transcripts e.g., cDNA or cRNA
  • one of skill in the art will understand how to design the complete set of capture oligonucleotides based on the teachings provided herein, e.g., in the Examples, below, and based on the complete set of oligonucleotides exemplified in Table I.
  • the oligonucleotide array comprises the capture oligonucleotides having the nucleic acid sequences set forth in Table IV, below.
  • the oligonucleotides in Table IV are examples of the oligonucleotides that can be designed to collectively target transcripts (e.g., cDNA or cRNA) encoding KIR polypeptides.
  • cDNA or cRNA collectively target transcripts
  • the oligonucleotide array comprises the capture oligonucleotides having the nucleic acid sequences set forth in Table V, below.
  • the oligonucleotides in Table V are examples of the oligonucleotides that can be designed to collectively target transcripts (e.g., cDNA or cRNA) encoding blood group determining molecules.
  • target transcripts e.g., cDNA or cRNA
  • B*53:ll B*53:12, B*53:13, B*53:14, B*53:15, B*53:16, B*53:17, B*53:18, B*53:19, B*53:20, B*53:21, B*53:22, B*53:23, B*54:01, B*54:02, B*54:03, B*54:04, B*54:06, B*54:07, B*54:09, B*54:10, B*54:ll, B*54:12, B*54:13, B*54:14, B*54:15, B*54:16, B*54:17, B*54:18, B*54:19, B*54:20, B*54:21, B*54:22, B*54:23, B*55:01:01, B*55:01:02, B*55:01:03, B*55:01:04, B*55:01:05, B*55:01:06, B*55
  • PSMA8_variantl PSMA8_variant2, PSMA8_variant3, PSMB1, PSMB2, PSMB3, PSMB4, PSMB5_variantl, PSMB5_variant2, PSMB5_variant3, PSMB6, PSMB7, PSMB8_variantl, PSMB8_variant2, PSMB9, PSMB10, PSMB11, PSMC1, PSMC2, PSMC3, PSMC4_variantl, PSMC4_variant2, PSMC5, PSMC6, PSMDl_variantl, PSMDl_variant2, PSMD2, PSMD3, PSMD4, PSMD5, PSMD6, PSMD7, PSMD8, PSMD9,
  • PSMD10_variantl PSMD10_variant2, PSMD11, PSMD12_variantl, PSMD12_variant2, PSMD13_variantl, PSMD13_variant2, and PSMD14.
  • the oligonucleotide array comprises the capture oligonucleotides having the nucleic acid sequences set forth in Table X, below, or the oligonucleotide array comprises the oligonucleotides in Table X that have names beginning with "HPT", “HPE” or “HPN” as shown in Table X.
  • Table X oligo names start with "HNN”, “HPN”, “HPE” or "HPT.”
  • “HNN” indicates that the oligo is a negative control oligonucleotide.
  • HPT indicates a "normal” (unchanged) oligo
  • HPT indicates a “truncated” oligo
  • HPE indicates an "extended” oligo.
  • the “HPT” and “HPE” modifications were made to ensure proper melting temperature.
  • the HPT”, “HPE” or “HPN” oligios in Table X are designed to collectively target transcripts (e.g., cDNA or cRNA) encoding a subset of clinically relevant HLA molecules as well as their 5'UTR regions.
  • the Table also contains negative control oligos, which help better identification of thresholds for positive and negative signals.
  • HLA alleles targeted by the oligos in Table X include: A*01:01:01:01, A*01:01:01, A*01:01:02, A*01:01:03, A*01:01:04, A*01:01:05, A*01:01:06, A*01:01:07, A*01:01:08, A*01:01:09, A*01:01:10, A*01:01:ll, A*01:01:12, A*01:01:13, A*01:01:14, A*01:01:15,
  • B*15:ll:04 B*15:ll:05, B*15:12, B*15:13:01, B*15:13:02, B*15:14, B*15:15, B*15:16:01 B*15:16:02, B*15:16:03, B* 15: 17:01 :01, B*15:17:01:02, B*15:17:02, B*15:18:01 B*15:18:02, B*15:18:03, B*15:18:04, B*15:20, B*15:21, B*15:23, B*15:24, B*15:25:01, B*15:25:02, B*15:25:03, B*15:27:01, B*15:27:02, B*15:27:03, B*15:29, B*15:30, B*15:31, B*15:32, B*15:34, B*15:35, B*15:37, B*15:38:01,
  • DRB1*04: 10 DRB1*04: 100, DRB1*04:101, DRB1*04:102, DRB1*04: 11, DRB1*04: 12, DRB1*04: 13, DRB1*04: 14, DRB1*04: 15, DRB1*04: 16, DRB 1*04: 17:01, DRB1*04: 17:02, DRB1*04: 18, DRB1*04: 19, DRB1*04:20, DRB1*04:21, DRB1*04:22, DRB1*04:23, DRB1*04:24, DRB1*04:25, DRB1*04:26, DRB1*04:27, DRB1*04:28, DRB1*04:29, DRB1*04:30, DRB1*04:31, DRB1*04:32, DRB1*04:33, DRB1*04:34, DRB1*04:35,
  • DRA negative control DRB negative control
  • DQA negative control DRA negative control
  • DQB negative control DPA negative control
  • DPB negative control DQB negative control
  • the oligonucleotide array comprises one or more of the negative control oligonucleotides having the nucleic acid sequences set forth in Table VI, below.
  • the oligonucleotides in Table VI are examples of the negative control oligonucleotides for classical and non-classical HLA molecules, that can be designed according to the methods described herein. As stated above, one of skill in the art will understand how to design the complete set of negative control oligonucleotides based on the teachings provided herein.
  • the oligonucleotide array comprises one or more of the negative control oligonucleotides having the nucleic acid sequences set forth in Table VII, below.
  • the oligonucleotides in Table VII are examples of the negative control oligonucleotides for accessory molecules that can be designed according to the methods described herein.
  • the oligonucleotide array comprises one or more of the negative control oligonucleotides having the nucleic acid sequences set forth in Table VIII, below.
  • the oligonucleotides in Table VIII are examples of the negative control oligonucleotides for KIR molecules that can be designed according to the methods described herein..
  • the oligonucleotide array comprises one or more of the negative control oligonucleotides having the nucleic acid sequences set forth in Table IX, below.
  • the oligonucleotides in Table IX are examples of negative control oligonucleotides for blood group determining molecules that can be designed according to the methods described herein.
  • the oligonucleotide array comprises one or more of the negative control oligonucleotides having the nucleic acid sequences set forth in Table X and denoted by "HN " in Table X, below, which are examples of such control oligonucleotides that can be designed according to the methods described herein.
  • oligo names begin with "HNN,” “HPN,” “HPE,” or “HPT.”
  • HNN indicates that the oligo is a negative control oligonucleotide
  • HPN indicates a "normal” (unchanged) oligo
  • HPT indicates a "truncated” oligo
  • HPE indicates an "extended” oligo. The “HPT” and “HPE” modifications were made to ensure proper melting temperature.
  • Table II-X The total number of oligonucleotides in each of the full sets shown partially in Tables II-X are as follows: 7,857 (Table II), 69,355 (Table III), 16,854 (Table IV), 61,673 (Table V), 9, 185 (Table VI), 66,004 (Table VII), 1,411 (Table VIII), 45,647 (Table IX), 75,476 (Table X).
  • Table I Classical HLA Capture Oligonucleotides
  • CTCTCGGGGGCCCTGGCCCT 86 CCATGAGGTATTTCTTCACATCCGTGTCCCG 132 GCCCCGCTTCATCGCCGTGGG
  • CTGGGGACCCTGCGCGGCTACTA 353 ACACCATCCAGATAATGTATGGCTGCGACGTG 399 CTTCCTCCGCGGGTACCGGCAG
  • AGCAGAGATACACCTGCCATGTGCAGCATG 882 CCTCACCCTGAGATGGGAGCTGTCTTC 928 GTGGGCATCATTGCTGGCCTGGTTCTC

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Abstract

L'invention concerne des puces d'oligonucléotides pour le typage tissulaire (par exemple le typage de tissu HLA). Plus particulièrement, les puces sont des puces à haute résolution utiles pour des évaluations diagnostiques et la détermination de la compatibilité de transplant entre donneur/receveur.
PCT/US2011/065624 2010-12-16 2011-12-16 Puce d'oligonucléotides pour le typage tissulaire WO2012083240A2 (fr)

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CN108342457A (zh) * 2018-04-01 2018-07-31 佛山市顺德区辉锦创兴生物医学科技有限公司 Hla-b5801等位基因的检测试剂盒及其应用
WO2020004802A1 (fr) * 2018-06-29 2020-01-02 경북대학교 산학협력단 Procédé d'attribution de typage final d'antigène de leucocyte humain dans un typage d'antigène de leucocyte humain à au moyen d'un essai de sonde oligonucléotidique spécifique à une séquence
KR20200002164A (ko) * 2018-06-29 2020-01-08 경북대학교 산학협력단 서열-특이적 올리고뉴클레오티드 프로브 방법에 의한 인간백혈구항원 형별 검사에서 인간백혈구항원의 최종 형별 판정 방법
KR102131293B1 (ko) 2018-06-29 2020-07-07 경북대학교 산학협력단 서열-특이적 올리고뉴클레오티드 프로브 방법에 의한 인간백혈구항원 형별 검사에서 인간백혈구항원의 최종 형별 판정 방법
WO2020168282A1 (fr) * 2019-02-14 2020-08-20 Vanderbilt University Détection de l'antigène-a*32:01 leucocytaire humain en liaison avec la détermination d'une hypersensibilité médicamenteuse avec éosinophilie et symptômes systémiques (dress), et méthodes de traitement d'une infection bactérienne chez un sujet atteint de dress à médiation par la vancomycine
WO2021234172A1 (fr) * 2020-05-22 2021-11-25 University Of Ulster Test génétique pour prédire la réponse aux médicaments anti-tnf
CN113278687A (zh) * 2021-05-20 2021-08-20 广州医科大学附属第二医院 用于hla-b1502和hla-a2402基因型检测的试剂盒
CN113278687B (zh) * 2021-05-20 2023-11-24 广州医科大学附属第二医院 用于hla-b1502和hla-a2402基因型检测的试剂盒
GR20210100795A (el) * 2021-11-15 2023-06-13 Ανωνυμη Εταιρια Κυτταρικων Και Μοριακων Ανοσολογικων Εφαρμογων, Εκκινητες και συνθηκες ταυτοχρονης ενισχυσης των hla γονιδιων hla-a, hla-b και hla-drb1
CN117737233A (zh) * 2024-02-21 2024-03-22 北京医院 用于检测hla-a29等位基因的uap寡核苷酸、试剂盒和方法
CN117737233B (zh) * 2024-02-21 2024-06-07 北京医院 用于检测hla-a29等位基因的uap寡核苷酸、试剂盒和方法

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